Membrane curvature affects the stability and folding kinetics of bacteriorhodopsin

Biological membrane is crucial for the function, stability and folding of membrane proteins. By studying the stability and folding kinetics of bacteriorhodopsin (bR) in lipid vesicles with different sizes, here we report the influence of membrane curvature (vesicle size) on the stability and folding kinetics of bR. The results show that both the stability and folding kinetics of bR can be significantly changed when reconstituted into mimic membranes with different curvatures. The stability of bR decreases and unfolding rate of bR increases with the growth of vesicle size, i.e. decrease of membrane curvature. Our results suggest that it is possible to regulate the properties of membrane proteins by changing the curvature of membranes.     

TMA-DPH a fluorescent probe of membrane dynamics in living cells. How to use it in phagocytosis

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells--specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phagocytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Miltiparticle computer simulation of photosynthetic electron transport in the thylakoid membrane

Further developing the method for direct multiparticle modeling of electron transport in the thylakoid membrane, here we examine the influence of the shape of the reaction volume on the kinetics of the interaction of the mobile carrier with the membrane complex. Applied to cyclic electron transport around photosystem I, with account of the distribution of complexes in the membrane and restricted diffusion of the reactants, the model demonstrates that the biphasic character of the dark reduction of P700⁺ is quite naturally explained by the spatial heterogeneity of the system.     

The plasma-membrane internalization and recycling is enhanced in macrophages upon activation with gamma-interferon and lipopolysaccharide; a study using the fluorescent probe trimethylaminodiphenylhexatriene

The lipophilic fluorescent probe trimethylaminodiphenylhexatriene (TMA-DPH), previously used as a plasma membrane marker in membrane fluidity and exocytosis studies, was shown, to monitor the plasma-membrane internalization-recycling shuttle movement in cells. Using this approach we present here kinetic and dose-response data, which give evidence that the plasma membrane flow is enhanced in bone marrow macrophages from various mouse strains, upon in vitro activation with gamma interferon (IFN-gamma) or bacterial lipopolysaccharide (LPS), within physiological dose ranges. The effect studied evolved in line with the usual development kinetics of macrophage activation. Complementary assays on membrane fluidity, surface charge density and membrane surface indicated no related changes. From these experiments it is concluded that the observed enhancement of the plasma membrane traffic does not originate from specific limited membrane modifications, but is merely a particular feature of the overall macrophage activation.     

细胞膜表面单分子事件的实时观察

单分子实时检测以高分辨率显微镜及纳米操作技术为支撑,实现在微观世界单分子水平探索和阐明生命现象的本质,进而展现生命科学的全景。本文综述了近年来单分子成像和检测领域的技术进展,以及将这些技术和创新方法在活细胞膜表面单分子事件研究中的应用,如蛋白质扩散、蛋白折叠动力学、膜受体装配及近膜区囊泡运动等。     

The kinetics of monazomycin-induced voltage-dependent conductance. I. Proof of the validity of an empirical rate equation

Monazomycin (a positively-charged, polyene-like antibiotic) induces a strongly voltage-dependent conductance in thin lipid membranes when added to one of the bathing solutions. We show here that the kinetics of conductance changes after a step of membrane potential are only superficially similar to the kinetics of the potassium gating system of squid giant axons, in that the beginning of conductance increases are growth functions of the time, as opposed to power functions of the time. We find that the rate constant (reciprocal of the time constant) of the growth varies with the approximately 2.6 power of the monazomycin concentration. The rate constant also varies exponentially with membrane potential such that an e-fold change is associated with a 10-11 mV change of membrane potential. We show that solutions of a simple differential equation are able to reproduce the actual conductance changes almost exactly. In the accompanying paper (Muller and Peskin. 1981. J. Gen. Physiol. 78:201-229), we derive the differential equation from a molecular model and use the theoretical equation so obtained to investigate the gating current of this system and to predict an interesting form of memory.     

Prediction of membrane protein types based on the hydrophobic index of amino acids

A new algorithm to predict the types of membrane proteins is proposed. Besides the amino acid composition of the query protein, the information within the amino acid sequence is taken into account. A formulation of the autocorrelation functions based on the hydrophobicity index of the 20 amino acids is adopted. The overall predictive accuracy is remarkably increased for the database of 2054 membrane proteins studied here. An improvement of about 13% in the resubstitution test and 8% in the jackknife test is achieved compared with those of algorithms based merely on the amino acid composition. Consequently, overall predictive accuracy is as high as 94% and 82% for the resubstitution and jackknife tests, respectively, for the prediction of the five types. Since the proposed algorithm is based on more parameters than those in the amino acid composition approach, the predictive accuracy would be further increased for a larger and more class-balanced database. The present algorithm should be useful in the determination of the types and functions of new membrane proteins. The computer program is available on request.     

A comparison of sodium channel kinetics in the squid axon,the frog node and the frog node with BTX using the “silent gate” model

In this paper it is shown that the very different kinetics measured for the rise of the sodium current which follows a depolarization of the membrane in the squid giant axon, the frog node and the frog node treated with Batrachotoxin may be accurately predicted using only the measured equilibrium and static characteristics for the three preparations and the kinetics measured for the gating charge transfer.The kinetic predictions follow the use of the silent gate model for ion channel gating. The model is electrostatic and its chief assumptions are that the channel gate, called here the N-system, has fast kinetics and responds to the gating charge that transfers but not directly to the trans-membrane voltage applied. Because channel gating, corresponding here to the motion of the N-system, does not change its energy in the trans-membrane applied electric field the gating is electrically silent as far as gating charge transfer measurement is concerned. However the probability of gating rises with the quantity of gating charge that transfers due to the electrostatic interaction between the N-system and the gating charge, redistributed under the influence of the applied trans-membrane electric field. With these assumptions the kinetics of sodium channel gating are predictable using only the static and equilibrium characteristics of gating charge and channel activation measured as a function of membrane voltage, and the kinetics of the gating charge transfer. Because of the fast kinetics assumed for the N-system the predicted kinetics are the same for channels with any number of equivalent and independent N-systems or gates acting in parallel.The model predictions for sodium permeability kinetics are compared in detail with those recently measured for the frog node treated with Batrachotoxin and excellent agreement is obtained.     

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABA_A receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50–200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABA_A receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABA_A receptor activity and their effects on a lipid bilayer model system containing cholesterol.     

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK-21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus-1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus-1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.     

A Model for the Interfacial Kinetics of Phospholipase D Activity on Long-Chain Lipids

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.     

Structure and dynamics of detergent-solubilized M13 coat protein (an integral membrane protein) determined by 13C and 15N nuclear magnetic resonance spectroscopy

The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection. M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies. An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments. The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei. Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics. A model is proposed for the structure of a coat protein dimer in detergent micelles.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

Characterisation of the R276A gain-of-function mutation in the ectodomain of murine P2X7

The cytolytic P2X7 purinoceptor is widely expressed on leukocytes and has sparked interest because of its key role in the activation of the inflammasome, the release of the pro-inflammatory cytokine IL-1β and cell death. We report here the functional characterisation of a R276A gain-of-function mutant analysed for its capacities to induce membrane depolarisation, calcium influx and opening of a large membrane pore permeable to YO-PRO-1. Our results highlight the particular sensitivity of R276A mutant to low micromolar adenosine triphosphate (ATP) concentrations, which possibly reflect an increased affinity for its ligands, and a slower closing kinetics of the receptor channel. Our findings support the notion that evolutionary pressures maintain the low sensitivity of P2X7 to ATP. We also believe that the R276A mutant described here may be useful for the generation of new animal models with exacerbated P2X7 functions that will serve to better characterise its role in inflammation and in immune responses.     

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.     

Membrane dynamics of the phosphatidylinositol-anchored form and the transmembrane form of the cell adhesion protein LFA-3.

The cell adhesion glycoprotein LFA-3 is expressed on the cell surface of nucleated cells in both a membrane-spanning form and a glycosyl phosphatidylinositol-anchored form. To determine whether distinct membrane anchors direct the dynamics of a given protein, the turnover of biosynthetically 35S-labeled and biotin surface-labeled LFA-3 molecules was followed. It is shown here that (a) expression of the two LFA-3 forms is regenerated with similar kinetics after enzymatic removal from the cell surface; (b) neither of the distinct LFA-3 molecules undergoes constitutive internalization; and (c) transmembrane and glycosyl phosphatidylinositol-anchored LFA-3 have an unusually long life span with an identical half-life of 50 h. Thus, the type of membrane anchor is not affecting turnover characteristics of a particular cell surface glycoprotein.